Cervical lymphoepithelioma-like carcinoma with deficient mismatch repair and loss of SMARCA4/BRG1: a case report and five related cases.

BACKGROUND: We encountered a cervical lymphoepithelial carcinoma (LEC) possessing a predominantly solid architecture with deficient mismatch repair (dMMR) and loss of expression of the SWI/SNF (SWItch/Sucrose Non-Fermentable) chromatin remodeling complex subunit. This is the first case report of LEC with dMMR and loss of SWI/SNF complex subunit. CASE PRESENTATION: A 34-year-old woman presented at our hospital with menstrual irregularities and abnormal vaginal bleeding. Magnetic resonance imaging revealed an exophytic mass in the posterior uterine cervix. Biopsy specimens confirmed squamous cell carcinoma with a 2018 International Federation of Gynecology and Obstetrics (FIGO) uterine cervical cancer stage of IB2. In a subsequent conization specimen, the tumor appeared exophytic. Microscopically, the tumor cells formed a predominant solid architecture. Abundant lymphocytic infiltration was observed. The pathological diagnosis indicated human papillomavirus (HPV)-associated squamous cell carcinoma with LEC pattern and pT1b2. Immunohistochemically, high programmed death-ligand 1 (PD-L1) expression, dMMR, and loss of the switch/sucrose non-fermentable family-related, matrix-associated, actin-dependent regulator of chromatin subfamily member 4 (SMARCA4)/BRG1, an SWI/SNF complex subunit, were observed. The patient underwent a radical hysterectomy and is alive without disease one year and five months later. Our analysis of five additional LEC cases revealed a consistent association with high-risk HPV and elevated PD-L1 expression. In addition to the present case, another patient exhibited dMMR. The SWI/SNF complex was retained except in the present case. The prognosis was favorable in all cases. CONCLUSIONS: This unique case of LEC with dMMR suggests a distinct clinical entity with potential immunotherapy implications. Analysis of the other five LEC cases revealed that LEC was immune hot, and immune checkpoint inhibitors may be effective. The two dMMR cases showed loss of MLH1 and PMS2 expressions, and prominently high tumor PD-L1 expression. In those cases, dMMR might have contributed to the morphological characteristics of LEC.

Generation of a tendon-like tissue from human iPS cells.

Tendons and ligaments are essential connective tissues that connect the muscle and bone. Their recovery from injuries is known to be poor, highlighting the crucial need for an effective therapy. A few reports have described the development of artificial ligaments with sufficient strength from human cells. In this study, we successfully generated a tendon-like tissue (bio-tendon) using human induced pluripotent stem cells (iPSCs). We first differentiated human iPSCs into mesenchymal stem cells (iPSC-MSCs) and transfected them with Mohawk (Mkx) to obtain Mkx-iPSC-MSCs, which were applied to a newly designed chamber with a mechanical stretch incubation system. The embedded Mkx-iPSC-MSCs created bio-tendons and exhibited an aligned extracellular matrix structure. Transplantation of the bio-tendons into a mouse Achilles tendon rupture model showed host-derived cell infiltration with improved histological score and biomechanical properties. Taken together, the bio-tendon generated in this study has potential clinical applications for tendon/ligament-related injuries and diseases.

Cytological features of BCOR-CCNB3 sarcoma: Comparison with Ewing sarcoma and synovial sarcoma.

INTRODUCTION: BCOR-CCNB3 sarcoma (BCS) is one of the histological types classified as an undifferentiated small round cell sarcoma of bone and soft tissue. This sarcoma frequently develops in males under 20 years of age. Histologically, a delicate capillary network has been reported as a conspicuous finding. In this study, the cytological findings of BCS were observed in two cases of primary lesions and one case of a lung metastatic lesion. The cytological findings of BCS were compared with its histological mimics, and the characteristic findings of BCS were examined. METHODS: Three cases of BCS were studied, and a cytological comparison was performed with 8 cases of Ewing sarcoma (ES) and 10 cases of synovial sarcoma (SS; monophasic type: 7 cases, biphasic type: 2 cases, poorly differentiated: 1 case). RESULTS: In all BCS cases, small clusters with thin and delicate vascular cores and tiny vascular fragments were conspicuous. In ES and SS cases, although small clusters with vascular cores were observed, the vascular cores were thicker than in BCS, and no tiny vascular fragments appeared in most cases. Cytomorphological differences of tumour cells were also observed among BCS, ES, and SS. Predominantly rounded nuclei with fine chromatin and inconspicuous nucleoli can be cytological clues for BCS. CONCLUSIONS: BCS shows characteristic cytological findings that make the diagnosis of BCS more likely than that of ES and SS. Cytological evaluation is a useful tool for appropriate differential diagnosis that leads to a more accurate final diagnosis and rapid treatment.

Isolation of balenine from opah (Lampris megalopsis) muscle and comparison of antioxidant and iron-chelating activities with other major imidazole dipeptides.

Balenine (Bal) in opah muscle was extracted using hot water and purified by ion-exchange chromatography and recrystallization to provide 41 g of over 95% pure Bal from 1 kg of opah muscle. The structure of purified Bal was identical to that of an authentic Bal standard by NMR analysis. The antioxidant (ORAC and HORAC values) and Fe(II) ion-chelating abilities of purified Bal were examined by comparison with two major imidazole dipeptides, carnosine (Car) and anserine (Ans). Opah-derived Bal showed significantly higher ORAC and HORAC values and Fe(II) ion-chelating ability at 0.3 mM. In silico molecular simulation revealed that Bal and Car formed hydrogen bonds between the hydrogen atom of the imidazole imino group and the carboxyl carbonyl oxygen, whereas Ans did not. The proposed method for extracting and purifying Bal from opah muscle suggests that opah can be utilized as a functional food or Bal resource.

Primary thymic adenocarcinoma with an aggressive clinical course: An autopsy case showing signet ring cell-like features.

Thymic adenocarcinoma is an extremely rare neoplasm, and little is known about its pathogenesis and clinical characteristics. A 52-year-old man presented to our clinic with severe dyspnea. At initial presentation, massive carcinomatous pleuritis and pericarditis were observed, and a lobulated mass in the anterior mediastinum was found on computed tomography. Cytological examination revealed adenocarcinoma accompanied by signet ring cells; however, his tumor showed aggressive growth without any possibility of treatment, and he died as a result of cancer progression within one month of admission. An autopsy confirmed thymic adenocarcinoma showing various histological features including mucinous, signet ring cell-like, and trabecular features. Immunohistochemically, the tumor cells were positive for cytokeratin (CK) (AE1/AE3) but negative for TTF-1. In addition, some tumor cells were positive for CD5 and KIT. Further examination revealed that tumor cells of the nonmucinous type were positive for CK7, and negative for CK20 and caudal-type homeobox 2 (CDX2). The tumor cells with mucinous and signet ring-like features were positive for CK20 and CDX2 and negative for CK7, indicating enteric differentiation. In particular, tumor cells with signet ring cell-like features indicated widespread lymphangitic carcinomatosis and pulmonary tumor thrombotic microangiopathy (PTTM). The presence of signet ring cell-like features with enteric differentiation is suggestive of a fulminant clinical course due to widespread lymphangiosis carcinomatosa and PTTM in patients with thymic adenocarcinoma. KEY POINTS: Thymic adenocarcinoma is an extremely rare neoplasm. Histological features of thymic adenocarcinoma include mucinous, signet ring cell-like, and trabecular features. Tumor cells with signet ring cell-like features indicate widespread lymphangitic carcinomatosis and pulmonary tumor thrombotic microangiopathy. The presence of signet ring cell-like features with enteric differentiation is associated with a fulminant clinical course.

Organizing Pneumonia after Nivolumab Treatment in a Patient with Pathologically Proven Idiopathic Pulmonary Fibrosis.

Acute exacerbation of pre-existing interstitial lung disease (ILD) associated with systemic anticancer therapy is recognized as a life-threatening adverse event of lung cancer treatment. Programmed cell death 1 (PD-1) checkpoint inhibitors, such as nivolumab, often induce pneumonitis in patients with cancer; however, the tolerance and safety of nivolumab for advanced lung cancer with ILD are unclear. We report a 72-year-old patient with lung cancer with pathologically proven idiopathic pulmonary fibrosis who was treated with nivolumab. She demonstrated pneumonitis with an organized pneumonia (OP) pattern, but no acute exacerbation of ILD featuring a diffuse alveolar damage (DAD) pattern. She was successfully treated with corticosteroid therapy, and maintained good disease control after the discontinuation of nivolumab. She also showed pseudoprogression of the primary tumor, implying infiltration of T-cells into the lung. These findings suggest that T-cell activation by nivolumab treatment might not be directly associated with acute ILD exacerbation, and that treatable OP might be a major pulmonary complication of nivolumab in patients with pre-existing ILD, similar to patients without underlying ILD.

Ablation of miR-146b in mice causes hematopoietic malignancy.

Excessive and constitutive activation of nuclear factor-κB (NF-κB) leads to abnormal cell proliferation and differentiation, leading to the development of malignant tumors, including lymphoma. MicroRNA 146a (miR-146a) and miR-146b, both of which carry an identical seed sequence, have been shown to contribute to inflammatory diseases and tumors by suppressing the expression of key molecules required for NF-κB activation. However, the functional and physiological differences between miR-146a and miR-146b in disease onset have not been fully elucidated. In this study, we generated miR-146b-knockout (KO) and miR-146a-KO mice by genome editing and found that both strains developed hematopoietic malignancies such as B-cell lymphoma and acute myeloid leukemia during aging. However, the B-cell lymphomas observed in miR-146a- and miR-146b-KO mice were histologically different in their morphology, and the malignancy rate is lower in miR-146b mice than miR-146a mice. Upon mitogenic stimulation, the expression of miR-146a and miR-146b was increased, but miR-146b expression was lower than that of miR-146a. Using a previously developed screening system for microRNA targets, we observed that miR-146a and miR-146b could target the same mRNAs, including TRAF6, and inhibit subsequent NF-κB activity. Consistent with these findings, both miR-146a- and miR-146b-KO B cells showed a high proliferative capacity. Taken together, sustained NF-κB activation in miR-146b KO mice could lead to the development of hematopoietic malignancy with aging.

Pembrolizumab and salvage chemotherapy in EGFR T790M-positive non-small-cell lung cancer with high PD-L1 expression.

Immuno-checkpoint inhibitors (ICI) have become an effective treatment option for non-small-cell lung cancer patients. However, ICI therapy was reported to be less effective in patients with epidermal growth factor receptor (EGFR) mutations than in those with wild-type EGFR. We report here that an non-small-cell lung cancer patient with the EGFR mutant T790M showed a programmed cell death ligand 1 (PD-L1) expression level that increased from <25% to >90% after eighth-line osimertinib therapy. He was treated with pembrolizumab as a ninth-line treatment, and attained stable disease. After the pembrolizumab therapy, he was treated with gemcitabine, which produced a good response despite being the 10th-line treatment. We should consider administering ICI and chemotherapy even to EGFR mutant patients after failure of EGFR tyrosine kinase inhibitor, especially in cases with high PD-LI expression.

Combinatorial CRISPR/Cas9 Approach to Elucidate a Far-Upstream Enhancer Complex for Tissue-Specific Sox9 Expression.

SRY-box 9 (SOX9) is a master transcription factor that regulates cartilage development. SOX9 haploinsufficiency resulting from breakpoints in a ∼1-Mb region upstream of SOX9 was reported in acampomelic campomelic dysplasia (ACD) patients, suggesting that essential enhancer regions of SOX9 for cartilage development are located in this long non-coding sequence. However, the cis-acting enhancer region regulating cartilage-specific SOX9 expression remains to be identified. To identify distant cartilage Sox9 enhancers, we utilized the combination of multiple CRISPR/Cas9 technologies including enrichment of the promoter-enhancer complex followed by next-generation sequencing and mass spectrometry (MS), SIN3A-dCas9-mediated epigenetic silencing, and generation of enhancer deletion mice. As a result, we could identify a critical far-upstream cis-element and Stat3 as a trans-acting factor, regulating cartilage-specific Sox9 expression and subsequent skeletal development. Our strategy could facilitate definitive ACD diagnosis and should be useful to reveal the detailed chromatin conformation and regulation.

MET amplification in endometrial cancers with clear-cell carcinoma components.

Endometrial clear-cell carcinoma (ECC) is relatively rare. The expression of diagnostic markers in this disease is similar to that of clear-cell carcinoma, but the molecular carcinogenic events and therapeutic targets are mostly unknown. MET gene amplification has been reported in various cancers, including ovarian clear-cell carcinomas; however, the MET gene status has not previously been examined in ECC. We performed real-time quantitative PCR (QPCR) and fluorescence in situ hybridization (FISH) to analyze the MET gene statuses of 12 ECC cases. We found MET amplifications in two cases (2/12; 16.7%) by both methods. Of the 12 cases, 9 were pure clear-cell carcinomas, and 3 were mixed types that included mixes with endometrioid carcinomas in 2 cases, and the remaining case was a heterologous-type carcinosarcoma that primarily consisted of a clear-cell carcinoma component and a scarce chondrosarcoma component. Both of the MET amplification cases were mixed; one contained endometrioid features, and the other chondrosarcoma features. This is the first report to analyze the statuses of the MET gene in ECCs, and the two mixed cases exhibited amplifications that are shared with ovarian clear-cell carcinomas. Further studies with larger numbers of cases are necessary to reveal the relationship between ECC and MET amplification.

A case of peritoneal malignant mesothelioma following radiation therapy for cervical cancer.

The present study presents a case of peritoneal malignant mesothelioma (PMM) following radiation therapy for cervical cancer. A 34-year-old Japanese woman, without asbestos exposure, was referred to the Department of Gynecologic Oncology, Saitama Medical University International Medical Center due to a cervical mass, and was diagnosed with cervical squamous cell carcinoma (SCC). The serum levels of tumor markers, including SCC antigen and cancer antigen 125 (CA125) were 229.0 ng/ml and 54.4 U/ml, respectively. The patient underwent concurrent chemoradiotherapy (CCRT), and a complete response was achieved. After 54 months, ascites was found at the rectouterine pouch, but peritoneal cytology suggested reactive mesothelial cell. After 62 months of CCRT, magnetic resonance imaging revealed masses in both the salpinges. The serum levels of SCC and CA125 were 0.9 ng/ml and 506.1 U/ml, respectively. Following this, left salpingectomy and peritoneal biopsy were performed laparoscopically. Histologic examination revealed atypical mesothelial cells with no continuity of background tubal epithelium. Immunohistochemistry showed positive staining for calretinin, thrombomodulin, mesothelin and glucose transporter 1. Based on these findings, the patient was diagnosed with PMM epithelioid type and underwent systemic chemotherapy; stable disease status has been obtained for 3 months. This case demonstrates the possibility of PMM occurrence within 10 years after radiotherapy, and indicates the importance of histological and immunohistochemical examination, particularly in cases of an atypical tumorigenesis pattern from the primary cancer.

Erratum: Pre-operative evaluation of circulating KL-6 levels as a biomarker for epithelial ovarian carcinoma and its correlation with tumor MUC1 expression.

[This corrects the article DOI: 10.3892/ol.2017.6254.].

Pre-operative evaluation of circulating KL-6 levels as a biomarker for epithelial ovarian carcinoma and its correlation with tumor MUC1 expression.

Krebs von den Lungen-6 (KL-6), a mucinous sialylated sugar chain on human mucin-1 glycoprotein (MUC1), is a diagnostic marker for interstitial lung diseases. Furthermore, elevated serum KL-6 levels have been observed in certain malignant tumor types of epithelial origin. The expression of MUC1 has been observed in patients with epithelial ovarian cancer (EOC) and is considered a potential therapeutic target. In the present study, KL-6 serum levels were investigated in patients clinically suspected of having malignant ovarian tumors. A total of 219 patients were enrolled in the study, which analyzed their serum KL-6 levels in addition to tumor expression of MUC1 using immunohistochemistry. High serum KL-6 levels were predominantly observed in patients with EOC, and did not occur in patients with benign or borderline tumors. The level of serum KL-6 was highly correlated with tumor stage, grade and histological type, and demonstrated superior sensitivity for the detection of ovarian cancer compared with that of serum cancer antigen 125. High serum KL-6 was significantly associated with shorter progression-free survival. In addition, tumor MUC1 expression status was significantly correlated with serum KL-6 levels. These data suggest that serum KL-6 may be a useful, non-invasive biomarker surrogate for tumor MUC1 expression in future clinical trials of MUC1-targeted therapy.

Correlation Between Tumor Mesothelin Expression and Serum Mesothelin in Patients with Epithelial Ovarian Carcinoma: A Potential Noninvasive Biomarker for Mesothelin-targeted Therapy.

BACKGROUND: The cell surface glycoprotein mesothelin is highly expressed in several malignant diseases. Normal mesothelin expression is limited to mesothelial cells lining the pleura, peritoneum, and pericardium, making it a biomarker and an attractive target for cancer therapy. METHODS: We investigated tumor mesothelin expression and serum mesothelin levels in patients with epithelial ovarian cancer or borderline tumors. In total, 161 patients selected from a previous prospective study were analyzed for tumor mesothelin expression using immunohistochemistry and serum mesothelin expression using enzyme-linked immunosorbent assay. RESULTS: Eighty-eight (68.8%) epithelial ovarian cancers and eight (24.2%) borderline tumors showed high mesothelin expression, which was associated with shorter progression-free and overall survival. The tumor mesothelin expression status was moderately correlated with serum mesothelin levels in epithelial ovarian cancer patients. Based on receiver operating characteristic analysis, a serum mesothelin level above 2.20 nM predicted high tumor mesothelin expression in epithelial ovarian cancer patients (area under the curve = 0.81). In 45 patients with recurrent epithelial ovarian cancer, we observed relatively lower levels of serum mesothelin, compared to the level at the primary diagnosis. We also tracked the change in the serum mesothelin level during the course of second-line chemotherapy and found a discrepancy between the clinical response and the serum mesothelin change in some patients, which suggested tumor heterogeneity among the tumor cells with or without mesothelin expression. CONCLUSION: Serum mesothelin may be a useful noninvasive biomarker surrogate for tumor mesothelin expression in future clinical trials for mesothelin-targeted therapy.

Complementary Critical Functions of Zfy1 and Zfy2 in Mouse Spermatogenesis and Reproduction.

The mammalian Y chromosome plays a critical role in spermatogenesis. However, the exact functions of each gene in the Y chromosome have not been completely elucidated, partly owing to difficulties in gene targeting analysis of the Y chromosome. Zfy was first proposed to be a sex determination factor, but its function in spermatogenesis has been recently elucidated. Nevertheless, Zfy gene targeting analysis has not been performed thus far. Here, we adopted the highly efficient CRISPR/Cas9 system to generate individual Zfy1 or Zfy2 knockout (KO) mice and Zfy1 and Zfy2 double knockout (Zfy1/2-DKO) mice. While individual Zfy1 or Zfy2-KO mice did not show any significant phenotypic alterations in fertility, Zfy1/2-DKO mice were infertile and displayed abnormal sperm morphology, fertilization failure, and early embryonic development failure. Mass spectrometric screening, followed by confirmation with western blot analysis, showed that PLCZ1, PLCD4, PRSS21, and HTT protein expression were significantly deceased in spermatozoa of Zfy1/2-DKO mice compared with those of wild-type mice. These results are consistent with the phenotypic changes seen in the double-mutant mice. Collectively, our strategy and findings revealed that Zfy1 and Zfy2 have redundant functions in spermatogenesis, facilitating a better understanding of fertilization failure and early embryonic development failure.

Serum folate receptor alpha as a biomarker for ovarian cancer: Implications for diagnosis, prognosis and predicting its local tumor expression.

Folate receptor alpha (FRA) is a GPI-anchored glycoprotein and encoded by the FOLR1 gene. High expression of FRA is observed in specific malignant tumors of epithelial origin, including ovarian cancer, but exhibits very limited normal tissue expression, making it as an attractive target for the ovarian cancer therapy. FRA is known to shed from the cell surface into the circulation which allows for its measurement in the serum of patients. Recently, methods to detect the soluble form of FRA have been developed and serum FRA (sFRA) is considered a highly promising biomarker for ovarian cancer. We prospectively investigated the levels of sFRA in patients clinically suspected of having malignant ovarian tumors. A total of 231 patients were enrolled in this study and analyzed for sFRA as well as tumor expression of FRA by immunohistochemistry. High sFRA was predominantly observed in epithelial ovarian cancer patients, but not in patients with benign or borderline gynecological disease or metastatic ovarian tumors from advanced colorectal cancers. Levels of sFRA were highly correlated to clinical stage, tumor grade and histological type and demonstrated superior accuracy for the detection of ovarian cancer than did serum CA125. High sFRA was significantly associated with shorter progression-free survival in both early and advanced ovarian cancer patients. Finally, tumor FRA expression status was strongly correlated with sFRA levels. Taken together, these data suggest that sFRA might be a useful noninvasive serum biomarkers for future clinical trials assessing FRA-targeted therapy.

Synchronous mucinous metaplasia and neoplasia of the female genital tract with external urethral meatus neoplasm: A case report.

•We present a case of multiple mucinous metaplasia and neoplasia of cervix, endometrium, fallopian tube, ovary, and mesenterium with external urethral meatus neoplasm.•Immunohistochemistry showed almost same pattern in each neoplasms.•PCR-direct sequencing showed no existence of both KRAS and GNAS mutations.•This report suggests a possibility of synchronous mucinous metaplasia and neoplasia "beyond" female genital tract.

Diffusion-weighted MR imaging findings of ovarian adenocarcinofibromas and adenofibromas.

We retrospectively evaluated pelvic magnetic resonance imaging including diffusion-weighted imaging (DWI) of 16 ovarian lesions (5 adenocarcinofibromas, 2 borderline adenofibromas, and 9 benign adenofibromas). All adenocarcinofibromas were detected as large solid areas of strong high signal on DWI, and seven of nine benign adenofibromas and both borderline adenofibromas demonstrated no areas of high signal or small areas of weak high signal. Solid components that appear as areas of strong high signal on DWI might represent a characteristic finding of adenocarcinofibromas.

Inulavosin and its benzo-derivatives, melanogenesis inhibitors, target the copper loading mechanism to the active site of tyrosinase.

Tyrosinase, a melanosomal membrane protein containing copper, is a key enzyme for melanin synthesis in melanocytes. Inulavosin inhibits melanogenesis by enhancing a degradation of tyrosinase in lysosomes. However, the mechanism by which inulavosin redirects tyrosinase to lysosomes is yet unknown. The analyses of structure-activity relationship of inulavosin and its benzo-derivatives reveal that the hydroxyl and the methyl groups play a critical role in their inhibitory activity. Intriguingly, the docking studies to tyrosinase suggest that the compounds showing inhibitory activity bind through hydrophobic interactions to the cavity of tyrosinase below which the copper-binding sites are located. This cavity is proposed to be required for the association with a chaperon that assists in copper loading to tyrosinase in Streptomyces antibioticus. Inulavosin and its benzo-derivatives may compete with the copper chaperon and result in a lysosomal mistargeting of apo-tyrosinase that has a conformational defect.

Identification of an AP2-family protein that is critical for malaria liver stage development.

Liver-stage malaria parasites are a promising target for drugs and vaccines against malaria infection. However, little is currently known about gene regulation in this stage. In this study, we used the rodent malaria parasite Plasmodium berghei and showed that an AP2-family transcription factor, designated AP2-L, plays a critical role in the liver-stage development of the parasite. AP2-L-depleted parasites proliferated normally in blood and in mosquitoes. However, the ability of these parasites to infect the liver was approximately 10,000 times lower than that of wild-type parasites. In vitro assays showed that the sporozoites of these parasites invaded hepatocytes normally but that their development stopped in the middle of the liver schizont stage. Expression profiling using transgenic P. berghei showed that fluorescent protein-tagged AP2-L increased rapidly during the liver schizont stage but suddenly disappeared with the formation of the mature liver schizont. DNA microarray analysis showed that the expression of several genes, including those of parasitophorous vacuole membrane proteins, was significantly decreased in the early liver stage of AP2-L-depleted parasites. Investigation of the targets of this transcription factor should greatly promote the exploration of liver-stage antigens and the elucidation of the mechanisms of hepatocyte infection by malaria parasites.